Aspartokinase of Lemna paucicostata Hegelm. 6746 · Plant Physiol. (1989) 90, 1577-1583 Receivedfor publication December31, 1988 0032-0889/89/90/1577/07/$01.00/0 AspartokinaseofLemnapaucicostata

Received for publication December 31, 1988Plant Physiol. (1989) 90, 1577-15830032-0889/89/90/1 577/07/$01 .00/0

Aspartokinase of Lemna paucicostata Hegelm. 6746

John Giovanelli*', S. Harvey Mudd, and Anne H. DatkoLaboratory of General and Comparative Biochemistry, National Institute of Mental Health,

Bethesda, Maryland 20892

ABSTRACT

A sensitive and specific method was developed for assay ofaspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicos-tata. Lysine inhibited approximately 93%, and threonine approx-imately 6%; together, these amino acids inhibited 99%. Inhibitionby lysine was synergistically increased by S-adenosylmethionine,which by itself had no effect on activity. Essentially completeinhibition of threonine-resistant activity was obtained with lysine,and of lysine-resistant activity with threonine. Inhibition by lysineand threonine was additive, with no indication of concerted inhi-bition. Aspartate concentration had no effect on the relativeproportions of lysine- and threonine-sensitive activities. Aspar-tokinase activity was in large excess of that reported by otherworkers, the maximum capacity (V,,,x) far exceeding the in vivorequirements. Estimations of rates of aspartokinase in vivo sug-gest that the step catalyzed by this enzyme may not be theoverall 'rate-limiting' one for entry of 4-carbon units into theaspartate family of amino acids, and that feedback inhibition ofthis enzyme by lysine and threonine may not be a major factor inregulating flux through this step.

(19, 25). However, the nature and extent of the regulation invivo remains to be clarified. In the present work we examinedaspartokinase of L. paucicostata, a higher plant possessingmany experimental advantages (9) and used in this laboratoryfor extensive studies on the regulatory patterns of biosynthesisof the aspartate family of amino acids (12, 13, 28). Wedeveloped a sensitive and specific assay suitable for crudeextracts to elucidate the salient catalytic and regulatory fea-tures of aspartokinase in this plant. This paper presents initialevidence that questions the validity of two tenets of currentschemes proposed for regulation of synthesis of the aspartatefamily of amino acids: (1) that aspartokinase is the overall'rate-limiting'3 step for entry of 4-carbon units into the aspar-tate family of amino acids (23, 25), and (b) that feedbackinhibition of aspartokinase by lysine and threonine is a majorfactor in regulating flux through this step ( 19).

MATERIALS AND METHODS

Chemicals

AdoMet (iodide salt, 85-90% pure) and L-aspartic acid-p-hydroxamate were obtained from Sigma Chemical Corp.

Aspartokinase (EC 2.7.2.4) catalyzes the first committingstep in the synthesis of the aspartate family of amino acids,lysine, methionine, threonine, and isoleucine (4, 19). In arecent review, Miflin and Lea (19) concluded that most plantscontain two aspartokinase activities, one inhibited by threo-nine, the other by lysine. The two forms from a variety ofplants have been physically separated (19, 23). The predomi-nant portion of the aspartokinase from most plants studied issensitive to lysine (4). The inhibition by lysine is synergisticwith AdoMet2 (24). Predominantly threonine-sensitive aspar-tokinase has been reported in peas, soybean, and Sinapis albaL. (4). The relative proportions can change also with thephysiologic state of the tissue; rapidly growing cells have ahigher proportion of lysine-sensitive activity than do older,nondividing cells (19). Aspartokinase ofLemna may be atyp-ical in that inhibition by lysine and threonine has beenclaimed to be concerted for the enzyme from L. minor (32).Initial reports of concerted inhibition by lysine and threonineof aspartokinase from wheat (31) and Cucumis (1) have notbeen substantiated by subsequent studies (3, 24, 33).

It has been proposed that aspartokinase plays a regulatoryrole in the biosynthesis of the aspartate family of amino acids

'Address correspondence to Building 36, Room 3D30, NationalInstitute of Mental Health, Bethesda, MD 20892

2Abbreviation: AdoMet, S-adenosylmethionine.1577

Solvents

Solvent A contained 2-propanol:88% HCOOH:H20 (7:1:2,v/v). Solvent B contained 95% ethanol:7.25 mM NH40H(90:10, v/v).

Buffer

Buffer A contained 25 mm K phosphate (pH 7.5), 2 mMEDTA, 2 mm MgCl2, 50 mM KCI, 2.0 M glycerol, and 30 mM2-mercaptoethanol.

Radioactive Compounds

L-[U-'4C]Aspartic acid (ICN Radiochemicals) and L-[2,3-3H]aspartic acid (Amersham) were purified by chromatogra-phy on Dowex 1. The radioactive compound was applied in10 mL of 20 mm pyridine formate (pH 5.9), to a column ofDowex 1 (0.8 x 2 cm) equilibrated with the same buffer. Thecolumn was washed with 5 mL of this buffer and labeledaspartate eluted with 20 mL of 0.1 M HCOOH. This solutionwas lyophilized to dryness and the residue dissolved in water.[3H]Aspartyl hydroxamate was prepared by incubation of [3H]

'As generally recommended (16, 20), the term 'rate-limiting'('pacemaker,' 'bottle neck,' etc.) is avoided, and is used here only forthe sake of accurate reporting of other workers' conclusions.

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Plant Physiol. Vol. 90, 1989

aspartic acid (585 nmol, 2.39 x 108 dpm) under the standardassay conditions of aspartokinase, modified by inclusion of0.4 M NH20H (K salt) in the reaction, and by increases inreaction time (to 4.3 h), temperature (to 30°C), and enzyme(to 116 ,ug of protein). [3H]Aspartyl hydroxamate was purifiedby Dowex 1 and paper chromatography as described for thestandard aspartokinase assay. Marker ['4Claspartate wasadded to the preparation prior to paper chromatography inorder to confirm resolution of aspartyl hydroxamate fromaspartate. The peak of [3H]aspartyl hydroxamate was elutedfrom the chromatogram in an overall yield of 20% from [3H]aspartate. The product cochromatographed in solvent A withauthentic L-aspartic acid-,B-hydroxamate visualized by reac-tion ofthe a-amino group with ninhydrin or the hydroxamategroup with FeCl3.

Plants

Lemna paucicostata Hegelm. 6746 was grown mixotroph-ically in medium 4 with 20 AM inorganic sulfate (9).

Preparation of Enzyme Extracts

All procedures were carried out at 0 to 4°C. Extracts wereprepared from fresh plants, since approximately one-third ofthe enzyme activity was lost in plants frozen at -80C over-night. Fronds (80-300) were homogenized in a glass homog-enizer with buffer A (approximately 1 ML/frond) containing1 mM L-lysine and 1 mM L-threonine. The homogenate wascentrifuged at 20,000g for 5 min and the supernatant fractionremoved with a Pasteur pipette. The pellet was suspended in100 ML of buffer A, recentrifuged as above, and the superna-tant solution combined with that from the original centrifu-gation. The combined supernatant fraction was gel filtered(28) with Sephadex G-25 (medium) equilibrated with bufferA. Gel filtration was repeated to minimize any transfer oflysine and threonine from the extraction medium to the assaymixture. Gel-filtered extracts were assayed immediately afterpreparation, since approximately one-half of the activity waslost after storage at -80C overnight. Protein was determinedas described (28).

Assay of Aspartate Kinase

When not specified either as threonine- or lysine-sensitive,'aspartokinase activity' refers to the total activity in bothforms.

Assay in Absence of NH20H (Standard Assay)

This assay was developed for specific and sensitive assay ofaspartokinase, and was used routinely unless stated otherwise.Plant extract was incubated with ['4Cjaspartate, resulting information of product [14C]aspartyl phosphate. After termi-nation ofenzyme activity, NH20H was added to convert [ 4C]aspartyl phosphate to the stable derivative, ['4C]aspartyl hy-droxamate. Aspartokinase activity was determined from theamount of "'C in this derivative after purification by chro-matography on Dowex 1 and by paper chromatography. Theassay contained the following components in a final volume

of 100 ,L; 6 mM L-[U-"'C]aspartate (2.3 x 106 dpm), 15 mMATP, 18 mM MgCl2, 1 mM DTT, 25 mM K phosphate, andenzyme extract containing up to 7 Mg of protein. The finalpH was 7.5. Incubation was for 5 min at 25C. Aspartylphosphate has a half-life of 150 min at 25°C and pH 7.7 to7.8 (26), so little chemical decomposition occurred during theincubation period. The assay was terminated by addition of45 ML of a solution containing 44 mm p-chloromercuriphen-ylsulfonic acid and 0.22 M EDTA. Incubation was then con-tinued for 5 min at 15sC to ensure complete inactivation ofthe enzyme. ["'C]Aspartyl phosphate was then converted to["'C]aspartyl hydroxamate by incubation for 20 min at 15°Cwith 100 uL of 1.7 M NH20H (K salt) (pH 7.5). Finally, thereaction was diluted with 1.0 mL of ice-cold H20. To analiquot of 0.4 mL of this solution was added 400 nmol ofcarrier aspartyl hydroxamate, authentic [3H]aspartyl hydrox-amate to serve as an internal marker, 32 ML of 1 N HCOOH,and 20 ML of 1 M pyridine fornate (pH 5.9); the final pH was5.9. The mixture was applied to a column of Dowex 1 (0.8 x5 cm) equilibrated with pyridine formate (pH 5.9), and thecolumn washed with 5 mL of 25 mm pyridine formate (pH5.9). Unreacted ['4C]aspartate was retained on the column.Aspartyl hydroxamate was contained in the combined flow-through and wash fractions. These were concentrated byevaporation, and chromatographed for 18 h with solvent A.Aspartyl hydroxamate migrated at RF 0.31, and was wellresolved from traces of [14C]aspartate (RF, 0.47). In rare in-stances (noted in the text) where resolution was not adequate,radioactive aspartyl hydroxamate was eluted from the chro-matogram and again chromatographed with solvent A. Theratio of 14C/3H in the aspartyl hydroxamate peak was used tocalculate the amount of aspartyl hydroxamate formed.

Assay in the Presence of NH20H

In this widely used assay, aspartokinase is determined bythe rate of formation of aspartyl hydroxamate in the contin-uing presence of NH20H. The assay is linear up to muchhigher concentrations ofprotein than is the aspartyl phosphateassay described above and was used for measurements of therelatively low activities of aspartokinase in the presence ofhighly inhibitory concentrations of lysine. The assay con-tained 0.4 M NH20H (K salt) in addition to the componentsof the standard assay. The reaction was terminated after 5min incubation at 25°C by addition of the p-chloromercuri-phenylsulfonic acid-EDTA mixture described above, followedby 1.0 mL of H20. An aliquot of 0.07 to 0.40 mL of thismixture, to which was added carrier and marker [3H]aspartylhydroxamate as described above, was titrated with 1 NHCOOH to approximately pH 5.9 (methyl red indicator).After addition of 20 ul of 1 M pyridine formate (pH 5.9), themixture was applied to a column ofDowex 1 and ['4C]aspartylhydroxamate determined as described for the standard assay.

RESULTS

Extraction of Aspartokinase

The presence of 1 mm lysine plus 1 mM threonine in theextraction medium increased recovery of total and lysine-

1 578 GIOVANELLI ET AL.



ASPARTOKINASE OF LEMNA PAUCICOSTATA HEGELM. 6746

sensitive aspartokinase activity by factors of 1.7- and 1.9-fold,respectively, without significantly affecting threonine-sensi-tive activity. The complete medium containing lysine plusthreonine was therefore used routinely for extraction of plants.Almost all (85%) of the recovered aspartokinase activity wasin the supernatant fraction.

Effects of Incubation Time, Enzyme Concentration, andNH20H on Aspartokinase Assay

The limits of incubation time and protein concentrationspecified for the standard assay should be exceeded only withextreme caution. In preliminary experiments in which proteinconcentrations and incubation times considerably exceededthose specified, the amount of product obtained decreasedwith increased enzyme and time of incubation. For example,after a 1 h incubation, the amount of aspartyl phosphateobtained with 130 ,ug of protein was only 15% that obtainedafter a similar incubation with 35 Ag of protein. As a result,the specific activity determined with the higher concentrationof protein was only 4% that with the lower. With furtherdecreases in enzyme and incubation time to those specifiedin the standard assay, the activity increased by another order.Within the limits defined in the standard assay, aspartylphosphate formation was linear with both time and enzymeconcentration. Activities of aspartokinase determined in thepresence of NH20H were identical to those determined withthe standard assay.

Catalytic Properties of Aspartokinase

Aspartokinase activity was completely dependent upon thepresence of enzyme and ATP. Omission of Mg2e reduced therate to less than 3% that of the standard assay. Figure 1 showsan Eadie-Hofstee plot of the results of experiments in whichaspartokinase activity was determined at increasing concen-trations of aspartate. The linear plot obtained in these limitedstudies is consistent with a hyperbolic relationship betweenvelocity and aspartate concentration, with an apparent Km foraspartate of 10 mM (SE, 0.8) and Vmax of 78 (SE, 5) nmol/min/mg protein.

Presence of Lysine- and Threonine-SensitiveAspartokinase Activities

Lysine inhibited aspartokinase activity by a maximum of80 to 94%, indicating the dominance of the lysine-sensitiveform of the enzyme. In determinations with extracts fromfour separate plant cultures, a mean value for maximal inhi-bition by lysine of93% (SE = 1.2, range = 89-94) was obtainedwith the standard aspartokinase assay, while a mean of 82%(n = 4, SE = 0.5, range = 80-83) was obtained for assaysperformed in the presence of NH2OH. The former value,indicating that the lysine-sensitive form comprises approxi-mately 90% of the total aspartokinase activity, is regarded asmore reliable; the possibility cannot be excluded that enzymesother than aspartokinase may catalyze the formation of smallamounts of aspartyl hydroxamate when NH20H is includedin the assay mixture (see "Discussion"). Percentage inhibitionsby lysine remained unchanged over a wide range (0.163,

70-

600

50

> 40

300

20

10

0~~~~~~~~~~~~~~~0 1 2 3 4 5 6 7

V/S

Figure 1. Effects of aspartate concentration on aspartokinase activ-ity-Eadie-Hofstee plot. Velocity (v) is in nmol/min/mg protein. Con-centration of aspartate (S) is in mm. The line of best fit was calculatedaccording to the method of Zivin and Waud (34). Reaction mixturesare those described for the standard assay, except for increasedamounts of 14C in aspartate, and changes in aspartate concentrations.The additional radioactivity in aspartate required that radioactiveaspartyl hydroxamate be subjected to rechromatography in solventA before determination of the ratio of 14C/3H.

0.663, 2.063, and 25.0 mM) of aspartate concentrations. Thre-onine caused approximately 20% maximal inhibition of totalaspartokinase activity, consistent with the minor contributionof the threonine-sensitive component.The effects of increasing concentrations of amino acid

inhibitor on each of the aspartokinase components are illus-trated in Figure 2. Both lysine- and threonine-sensitive en-zymes are strongly inhibited by their respective amino acidinhibitor, with 50% inhibition observed at approximately 40Mm lysine and 60 )lM threonine. Lysine-sensitive aspartokinaseapproached almost complete inhibition at concentrations oflysine above 1 mM; the threonine-sensitive form approachedan apparent maximum inhibition of approximately 90% atcomparable concentrations.

Inhibitions in the combined presence of lysine and threo-nine approximated the sum with either inhibitor alone, withno indication of cooperative inhibition by these amino acids.

Cooperative Effect of AdoMet on Lysine Inhibition

Figure 3 demonstrates the cooperative inhibition of aspar-tokinase by lysine and AdoMet. Concentrations of AdoMetalone up to 200 Mm had negligible effects on aspartokinaseactivity. By contrast, over the same range of concentrations,AdoMet caused progressive increases up to fourfold in theinhibition by 16 Mm lysine. No cooperative effects were ob-served with threonine and AdoMet.

1 579




0

~50

40

30

20

10

'0 0.5 1.0 1.5 5

LYSINE OR THREONINE, mM

Figure 2. Inhibition of lysine- and threonine-sensitive aspartokinasesby the respective amino acids. (0), Inhibition of lysine-sensitive as-partokinase by lysine. Inhibitions were calculated from the activitiesdetermined at each specified concentration of lysine (plus 5 mMthreonine) compared to that determined with 5 mm threonine alone.(A), Inhibition of threonine-sensitive aspartokinase by threonine. In-hibitions were calculated from the activities determined at eachspecified concentration of threonine (plus 5 mm lysine) compared tothat determined with 5 mm lysine alone. All assays were in thepresence of NH20H.

60

50

0

C._

0-

.0

c

40

30

20

10

0 le0

AdoMet ,uMFigure 3. Cooperative inhibition of aspartokinase by AdoMet andlysine. Percentage inhibition is plotted against increasing concentra-tions of AdoMet (A), or AdoMet plus 16 MM lysine (0).

DISCUSSION

A major problem in studies of aspartokinase in plants hasbeen the lack of a suitable assay of this enzyme in crudeextracts (10, 18). Neither of the two types of assays currently

in use is entirely satisfactory. In one, aspartyl phosphateproduction is coupled via aspartic semialdehyde dehydrogen-ase to NADH oxidation. This assay can be complicated byhigh background rates of NADH oxidation, and requiresisolation of the coupling enzyme and demonstration that it isnot affected by the presence of the effectors, etc. under study.In the other type of assay, aspartyl phosphate reacts withNH20H present in the reaction mixture to form the stablederivative, aspartyl hydroxamate. This assay suffers from thedisadvantages of possible modification of enzyme propertiesby high concentrations of NH20H (10) and in not beingspecific for aspartokinase. In the constant presence ofNH20Hin the assay, asparagine synthetase and aspartyl-t-RNA syn-thetase each catalyze synthesis of aspartyl hydroxamate viaan enzyme-bound aspartyladenylate intermediate. Both dis-advantages can be avoided by omission of NH20H from thereaction mixture until after termination of enzyme activity,as was done in the present work. This tactic has not beenused previously for routine determinations of aspartokinase,although it has occasionally been used for comparison withassays carried out in the presence of NH20H (10, 11). Thistactic, as well as the use of ['4C]aspartate of high specificactivity, provided an assay of extreme sensitivity, capable ofmeasuring rates as low as 100 pmol/min. The high sensitivitywas a critical factor in permitting the assay to be carried outat the low levels of protein and short incubation times whichyielded maximal specific activities.Both the total aspartokinase activity and the relative pro-

portions sensitive to lysine and threonine were of majorinterest in these studies. It was therefore important to provideoptimal conditions for recovery of these activities. Buffer Ahas been used successfully by Rognes and coworkers (23, 24)for isolation from a variety of plant tissues of aspartokinaseactivities sensitive predominantly to either lysine or threonine.In the present work, addition oflysine plus threonine to bufferA approximately doubled recovery of the major, lysine-sen-sitive aspartokinase activity without affecting recovery ofthre-onine-sensitive aspartokinase. The improved recovery prob-ably results from protection by lysine of lysine-sensitive as-partokinase, as reported for this enzyme from other planttissues (6, 22, 27).

Aspartokinase activity from L. paucicostata resembles thatisolated from a number of other plants (4, 19) in that a majorfraction (at least 80%) is inhibited by lysine, with most of theremaining activity being inhibited by threonine. The apparentKm (10 mM) for aspartate is similar to values reported foraspartokinases isolated from other tissues (18). The concen-trations of 40 ,uM lysine or 60 Mm threonine required for half-maximal inhibition of the respective lysine- and threonine-sensitive activities of L. paucicostata fall near the lower endsofthe ranges of 13 to 700 ,uM and 100 to 570 ,M, respectively,reported for aspartokinases isolated from other plants (4, 18).While having little effect alone, AdoMet appreciably increasedthe inhibition by lysine. No evidence was obtained for coop-erative inhibition by lysine and threonine, as has been claimedfor aspartokinase from L. minor (32).Based on the determined properties of the enzyme and

estimations of its cellular environment, calculations weremade of the maximum potential rates of aspartokinase in





Table I. V,., Values of Aspartokinase Relative to in vivo RequirementsTotal flux through aspartokinase in vivo (13.4 nmol/frond-doubling) was estimated from the combined amounts of protein lysine (4.5 nmol/

frond, ref. 14), protein methionine (1.0 nmol/frond, ref. 15), protein threonine (4.5 nmol/frond, Ref. 13), and protein isoleucine (3.4 nmol/frond,ref. 13) in L. paucicostata. Aspartokinase activities are reported at saturating concentrations of substrates (aspartate and ATP) and Mg2+. Lysine-or threonine-sensitive forms of aspartokinase are not assigned to any particular branch in the aspartate family, since our studies (13, 14) fail toindicate any channeling of the products of these two activities. Condition 1: Activity in the absence of inhibitors. The mean aspartokinase activityfor control cultures was 70 pmol/min/frond (n = 11, SE = 6, range 42-109) determined under standard assay conditions (6 mM aspartate, 15mM ATP, 18 mm Mg2+). From this value was calculated a Vw, of 187 pmol/min/frond based on an apparent Km of 10 mm for aspartate. It wasassumed that the concentrations of ATP and Mg2+ in the standard assay approached saturation; studies of aspartokinase in other plant tissuesshow the apparent Km of ATP ranges between 0.6 and 5 mm (19, 22), with optimal rates obtained at approximately equimolar concentrations ofMg2+. The latter aspartokinase activity was then converted to a value of 565 nmol/frond-doubling by multiplication by 60 x 24 x 2.1. The valueof 2.1 is the number of frond.d equivalent to a frond-doubling for control plants with a doubling time of 35 h, and was calculated as described(7). Condition 2: Activity in the presence of the physiological concentrations of 29 MM lysine (14), 190 Mm threonine (14), and 15 AM AdoMet (8)present in control cultures of L. paucicostata, assuming uniform distributions of these compounds throughout the plant. The value of 242 nmol/frond .doubling was calculated from the combined amounts of threonine-sensitive (6 nmol/frond-doubling) and lysine (plus AdoMet)-sensitive(236 nmol/frond -doubling) activities. Threonine-sensitive activity in the presence of 190 AM threonine was calculated by multiplying the threonine-sensitive aspartokinase activity of control plants (7% of 565 = 40 nmol/frond-doubling) by the fraction (15%) of this activity remaining at thisconcentration of threonine (Fig. 2). Activity of lysine-sensitive aspartokinase in the presence of 29 AM lysine (315 nmol/frond-doubling) wascalculated by multiplying lysine-sensitive activity of control plants (93% of 565 = 525 nmol/frond-doubling) by the percentage (from Fig. 2) ofapproximately 60% of this activity remaining in the presence of this concentration of lysine. The additional effect of 15 AM AdoMet was estimatedfrom the results of Figure 3, which indicate that activity in the presence of 15 AM AdoMet plus lysine would have been approximately 75% ofthat with lysine alone. Condition 3: Activity in the presence of estimated chloroplastic concentrations of lysine, threonine, and AdoMet.Chloroplastic concentrations of lysine (70 Mm) and threonine (750 AM) were approximated by multiplying the tissue concentrations of these aminoacids given in condition 2 by the ratio of the relative concentrations of the respective amino acid in chloroplasts and whole leaves of Zea mays(5). A value of 15 Mm AdoMet was assumed in the absence of data on the chloroplastic concentration of this compound. Aspartokinase activitieswere calculated essentially as described in condition 2. Condition 4: Complete inhibition of threonine-sensitive aspartokinase, without inhibitionof the lysine-sensitive form. Shown in parentheses are corresponding values that also include inhibition of the lysine-sensitive form by tissueconcentrations of 29 MM lysine and 15 AM AdoMet in control plants. Derivation of the resultant activity of 236 nmol/frond * doubling is given undercondition 2. Condition 5: Complete inhibition of lysine-sensitive aspartokinase, without inhibition of the threonine-sensitive form. Shown inparentheses are corresponding values that also include inhibition of the threonine-sensitive form by the tissue concentration of 190 AM threoninein control plants. Derivation of the resultant activity of 6 nmol/frond.doubling is given under condition 2. Condition 6: Complete inhibition oflysine- and threonine-sensitive aspartokinase. The value of 4 nmol/frond-doubling is based on the calculation that approximately 0.7% of thetotal aspartokinase appeared insensitive to inhibition by either lysine or threonine. The value of 0.7% was calculated from the expression:percentage of total activity resistant to inhibition by lysine (100-93 = 7%) x percentage (10%) of lysine-resistant activity resistant to threonineinhibition (Fig. 2). The value of 0.7% sets an upper limit, since it remains to be established conclusively that the extremely low activity in thepresence of lysine plus threonine can in fact be attributed to aspartokinase.

Condition Conditions of Measurement Aspartokinase ActivityNo.

nmol/frond-doubling -fold in vivo flux1 No inhibitors 565 422 Plus Lys, Thr, AdoMet (tissue concentrations) 242 183 Plus Lys, Thr, AdoMet (chloroplast concentrations) 118 94 Complete inhibition of Thr-sensitive 525 (236) 40 (18)5 Complete inhibition of Lys-sensitive 40 (6) 3 (0.45r6 Complete inhibition of Lys- and Thr-sensitive 4 0.3

The value of 0.45 was calculated as described in the legend on the basis of the estimated combined in vivo flux of 13.4 nmol/frond * doublinginto protein lysine, methionine, threonine, and isoleucine. Tissue concentrations of lysine resulting in complete inhibition of lysine-sensitiveaspartokinase would cause extensive feedback inhibition of lysine biosynthesis (14), thereby reducing the estimated in vivo flux throughaspartokinase to approximately 13.4-4.5 = 8.9 nmol/frond doubling. Based on this value, threonine-sensitive aspartokinase would provide 6/8.9 = 0.67-fold the in vivo flux.

vivo. These estimates (Table I) indicate that the maximum ments (condition 4). Even the Vmax of the minor, threonine-capacity (Vm,) of aspartokinase far exceeds the in vivo re- sensitive activity approximates the in vivo requirements (con-quirement for this enzyme. The large potential excess capacity dition 5). Only as complete inhibition of both lysine- andof aspartokinase pertains not only in the absence of inhibitors threonine-sensitive components is approached (condition 6)(condition 1), but also with the concentrations of lysine and was the Vma, of aspartokinase reduced below that required forthreonine (and AdoMet) determined in whole tissues (condi- in vivo flux for combined synthesis of the aspartate familytion 2), and estimated in chloroplasts (condition 3), the or- amino acids. Together, these finding suggest that althoughganelle in which aspartokinase is localized (29, 30). The results lysine and threonine may normally inhibit aspartokinase ac-of Table I further show that the V.,. for lysine-sensitive tivity, such inhibition is not severe enough to make asparto-aspartokinase acting alone is in large excess of in vivo require- kinase activity limiting, i.e. feedback inhibition by lysine and

1581




threonine may not normally be a major factor in regulatingflux through the aspartokinase step. Confirmation of thesesuggestions is provided in the companion paper (14) whichpresents definitive in vivo studies involving measurements ofthe actual fluxes through the aspartokinase step and into eachof the aspartate family of amino acids under a variety ofgrowth conditions.To what extent are these findings relevant to other higher

plants? Aspartokinase activities reported for other plants aretypically an order of magnitude less than those observed herefor L. paucicostata,4 and less than half the activity of anyother enzyme in the aspartate pathway for which assays areavailable (25). On the basis of the latter observation, Rogneset al. (25) tentatively concluded that aspartokinase is theoverall 'rate-limiting' step for entry of 4-carbon units fromaspartate into the aspartate family of amino acids. We believethat the reported activities of aspartokinase may have appre-ciably underestimated the maximum in vivo capacity of thisenzyme,5 and that the proposed role of aspartokinase inregulating entry of 4-carbon units into the aspartate family ofamino acids should be reevaluated. In agreement with thispossibility, in spite of the apparently low aspartokinase activ-ity reported for excised barley embryos, the ability of thistissue to grow at a normal rate with a concentration of lysineexpected to cause severe inhibition of aspartokinase in vivo(25) strongly suggests that this enzyme normally operatesbelow its maximum capacity, and that lysine is not a majorregulator in vivo of flux through the aspartokinase step. Afurther example is given by studies of Yamada et al. (33) withwheat cell cultures. The aspartokinase activity of 3.6 nmol/min/mg protein reported for this tissue is typical of the lowvalues reported for most plant tissues. However, as pointedout by Yamada et al. the ability of cultures supplementedwith lysine to grow at a normal rate shows that "considerableinhibition of aspartokinase by lysine does not affect its abilityto provide precursors required for the biosynthesis of otheraspartate-derived amino acids." An additional problem inassessing the physiological significance of previously reportedaspartokinase activities is that quantitative data are rarelyavailable to permit comparison of these activities with therequired in vivo fluxes. A review ofthe literature revealed only

'The mean specific activity of aspartokinase determined withcrude extracts of L. paucicostata (38 nmol/min/mg protein, n = 10;SE =3, range = 19-53) was compared with reported values determinedunder similar assay conditions for crude extracts (sometimes sub-jected to preliminary purification) from other plant tissues. Of a totalof 13 different tissues for which values were available (1, 11, 17, 18,21, 24, 32), only one was reported to have a specific activity ofaspartokinase greater than 12% that of L. paucicostata: aspartokinasefrom pea leaves purified to an unspecified extent with DEAE-cellulose(17) had an activity of 24% that of L. paucicostata.

IA number of reasons may be proposed why previously reportedactivities of aspartokinase may have underestimated in vivo activities.Results presented in this work show that appreciable activity can belost during isolation (in the absence of lysine plus threonine) andstorage of the enzyme. Furthermore, the low and variable activitiesdetermined in preliminary experiments (see "Results") emphasize thecritical importance of optimization of the particular assay conditions(especially time of incubation and enzyme concentration) beingemployed.

one example that allowed such direct comparison. Interest-ingly, this study (2) showed that after 5 d growth of carrot cellsuspension cultures the extracted aspartokinase activity wasapproximately 3-fold the in vivo requirement, and increasedto more than 60-fold after 18 d growth. These limited obser-vations suggest that higher plants in general may containexcess capacity of aspartokinase, and that this enzyme step isnot a major site for feedback regulation of flux.

LITERATURE CITED

1. Aarnes H (1974) Aspartate kinase from some higher plants andalgae. Physiol Plant 32: 400-402

2. Bright SWJ, Leggatt MM, Miflin BJ (1979) Amino acid levelsin carrot cell suspension culture. No correlation with aspartatekinase isoenzyme levels. Plant Physiol 63: 586-588

3. Bright SWJ, Shewry PR, Miflin BJ (1978) Aspartate kinase andthe synthesis of aspartate-derived amino acids in wheat. Planta139:119-125

4. Bryan JK (1980) Synthesis of aspartate family and branched-chain amino acids. In BJ Miflin, ed, The Biochemistry ofPlants, Vol. 5. Academic Press, New York, pp 403-452

5. Chapman DJ, Leech RM (1979) Changes in pool sizes of freeamino acids and amides in leaves and plastids of Zea maysduring leaf development. Plant Physiol 63: 567-572

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Aspartokinase of Lemna paucicostata Hegelm. 6746 · Plant Physiol. (1989) 90, 1577-1583 Receivedfor publication December31, 1988 0032-0889/89/90/1577/07/$01.00/0 AspartokinaseofLemnapaucicostata